E. V. Kulakovskaya

ATP leakage from yeast cells treated by extracellular glycolipids of Pseudozyma fusiformata

The ustilaginaceous yeast Pseudozyma fusiformata secreted glycolipids which were lethal to many yeasts and fungi more active at pH of about 4.0, and in the temperature range of 20-30 degrees C. Purified glycolipids enhanced non-specific permeability of the cytoplasmic membrane in sensitive cells, which resulted in ATP leakage and susceptibility of the cells to staining with bromocresol purple. Cells of Saccharomyces cerevisiae lost the ability to acidify the medium. Basidiomycetous yeasts were more sensitive to the glycolipids than ascomycetous ones. The minimal effective glycolipid concentration was 0.13 and 0.26 mg ml(-1) for Cryptococcus terreus and Filobasidiella neoformans, while for Candida albicans and Saccharomyces cerevisiae it was 1.0 and 1.6 mg ml(-1).

Inactivation of PPX1 and PPN1 genes encoding exopolyphosphatases of Saccharomyces cerevisiae does not prevent utilization of polyphosphates as phosphate reserve

Cytosol polyphosphates (polyPs) are the main phosphate (Pi) reserve in the yeast Saccharomyces cerevisiae. In this work, the participation of cytosol polyPs and exopolyphosphatases in maintenance of Pi homeostasis under Pi deficit in the cultivation medium has been studied in different strains of S. cerevisiae. The growth of yeast strains with inactivated genes PPX1 and PPN1 encoding the yeast exopolyphosphatases and a strain with double mutations in these genes in a Pi-deficient medium is not disturbed. All the studied strains are able to maintain relatively constant Pi levels in the cytosol. In Pi-deficient medium, polyP hydrolysis in the cytosol of the parent and PPN1-deficient strains seems to be performed by exopolyphosphatase Ppx1 and proceeds without any change of the spectrum of polyP chain lengths. In the PPX1-deficient strain, long-chain polyPs are depleted first, and only then short-chain polyPs are hydrolyzed. In the double PPX1 and PPN1 mutant having low exopolyphosphatase activity, polyP hydrolysis in the cytosol starts with a notable delay, and about 20% of short-chain polyPs still remain after the polyP hydrolysis in other strains has almost been completed. This fact suggests that S. cerevisiae possesses a system, which makes it possible to compensate for inactivation of the PPX1 and PPN1 genes encoding exopolyphosphatases of the yeast cells.

Antifungal cellobiose lipid secreted by the epiphytic yeast Pseudozyma graminicola

The yeast Pseudozyma graminicola isolated from plants inhibited growth of almost all ascomycetes and basidiomycetes tested (over 270 species of ca. 100 genera) including pathogenic species. This yeast secreted a fungicidal agent, which was identified as a glycolipid composed of cellobiose residue with two O-substituents (acetyl and 3-hydroxycaproic acid) and 2,15,16-trihydroxypalmitic acid. The release of ATP from the glycolipid-treated cells indicated that this glycolipid impaired the permeability of the cytoplasmic membrane. Basidiomycetes were more sensitive to the cellobiose lipid than ascomycetes.

Fungicidal activity of cellobiose lipids from culture broth of yeast Cryptococcus humicola and Pseudozyma fusiformata

Cellobiose lipids of yeast fungi Cryptococcus huminola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.

Efflux of potassium ions from cells and spheroplasts of Saccharomyces cerevisiae yeast treated with silver and copper ions

Silver ions induce the efflux of potassium from cells of the yeast Saccharomyces cerevisiae but have no such effect on spheroplasts. Copper ions and the natural fungicide 2-O-3-hydroxyhexanoyl-β-D-glucopyranosyl-(1→4)-(6-O-acetyl-β-D-glucopyranosyl-(1→16)-2,15,16-trihydroxyhexadecanoic acid) induce the efflux of potassium ions from both cells and spheroplasts of S. cerevisiae. Silver and copper ions inhibit the activity of the plasma membrane H+-ATPase during the treatment of both cells and spheroplasts. It is supposed that the inability of silver ions to stimulate potassium efflux from spheroplasts results from damage to some components of K+ transport systems during preparation of spheroplasts.

Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel

Summary Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-β-D-GlcpA and pentasaccharide β-D-Xylp-(1→3)-β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[β-D-Quip-(1→4)]-β-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.

The Fungicidal Activity of an Extracellular Glycolipid from Sympodiomycopsis paphiopedili Sugiyama et al.

The yeast Sympodiomycopsis paphiopedili (Ustilaginomycetes) produces an extracellular glycolipid, which possesses the maximum antifungal activity at a pH of the medium equal to 4.0–4.5. Among the approximately 300 tested species of yeastlike and mycelial fungi, more than 80% (including species pathogenic for plants, animals, and humans) were found to be sensitive to this glycolipid.

Study of the content of inorganic polyphosphates in Saccharomyces cerevisiae grown on different carbon sources with different O2 concentrations in the medium

The content of different fractions of inorganic polyphosphates (polyP) was studied in Saccharomyces cerevisiae VKM Y-1173 growing on a complete medium with glucose under hypoxia and active aeration as well as on ethanol. The highest growth rate was observed for aerobic fermentation, while the yield of biomass was maximal for cultivation on ethanol. In the mid-log growth phase, the amount of poly P was maximal in the cells grown on glucose under hypoxia and minimal on ethanol. In this latter case, the content of different poly P fractions changed unevenly: polyP3, polyP4, and polyP1 decreased by approximately 60%, 45%, and 30%, respectively; the salt-soluble polyP2 remained at almost the same level; while polyP5 abruptly increased 10-to 15-fold. These findings demonstrate that the metabolic pathways for poly P fractions are different. A significant drop in the amount of the main poly P fractions accompanied by a decrease of the poly P average chain length in the presence of carbon and Pi sources in the medium is evidence of active involvement of poly P as additional energy sources in the flows of energy in actively growing yeast cells.

Polyphosphates and exopolyphosphatases in cytosol and mitochondria of Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus.

Content and chain lengths of inorganic polyphosphates (polyP) as well as exopolyphosphatase activities were compared in cytosol and mitochondria of the yeast Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus. PolyP metabolism in cytosol and mitochondria was substantially dependent upon the carbon source. Acid-soluble polyP accumulated mainly in cytosol using either glucose or ethanol. The level of the accumulation was lower during growth on ethanol compared to that on glucose. Increase in polyP content in mitochondria was observed during growth on glucose, but not on ethanol. In cytosol the activity of exopolyphosphatase PPN1 was increased and the activity of exopolyphosphatase PPX1 was decreased independently of the carbon source under phosphate surplus conditions. Growth on ethanol caused exopolyphosphatase PPN1 to appear in the soluble mitochondrial fraction, while during growth on glucose only exopolyphosphatase PPX1 was present in this fraction.

Effects of cellobiose lipid B on Saccharomyces cerevisiae cells: K+ leakage and inhibition of polyphosphate accumulation

Cellobiose lipid B, a natural fungicide produced by the yeast Pseudozyma fusiformata, induces the leakage of K+ and ATP from cells of Saccharomyces cerevisiae. The presence of glucose decreases the effective concentration of cellobiose lipid B. The concentration of cellobiose lipid B was selected that results in a high rate of K+ leakage and a five-to sevenfold decrease in the intracellular ATP content, while the accumulation of acid-soluble polyphosphates decreased only by half. These results indicate the possibility of synthesis of these polymers which is independent of the ATP level and of the ion gradient on plasma membranes.